Glaucoma is one of the leading causes of blindness. Approximately 15% of cases of blindness world-wide result from glaucoma. The most common type, primary open-angle glaucoma, has a prevalence of 1/200 in the general population over 40 years of age. Glaucoma has been simply defined as the process of ocular tissue destruction caused by a sustained elevation of the Intra Ocular Pressure (IOP) above its normal physiological limits. Although several etiologies may be involved in the glaucoma complex, an absolute determinant in therapy selection is the amount of primary and/or induced change in pressure within the iridocorneal angle.
Current therapies include medications or surgeries aimed at lowering this pressure, although the pathophysiological mechanisms by which elevated IOP leads to neuronal damage in glaucoma are unknown. Medical suppression of an elevated IOP can be attempted using four types of drugs: (1) the aqueous humor formation suppressors (such as carbonic anhydrase inhibitors, beta-adrenergic blocking agents, and alpha2-adrenoreceptor agonists); (2) miotics (such as parasympathomimetics, including cholinergics and anticholinesterase inhibitors); (3) uveoscleral outflow enhancers; and (4) hyperosmotic agents (that produce an osmotic pressure gradient across the blood/aqueous barrier within the cilliary epithelium). A fifth category of drugs, neuroprotection agents, is emerging as an important addition to medical therapy, including, for example, NOS inhibitors, excitatory amino acid antagonists, glutamate receptor antagonists, apoptosis inhibitors, and calcium channel blockers.
Reviews of various eye disorders and their treatments can be found in the following references: Bunce et al., 2005, Associations between the deletion polymorphism of the angiotensin 1-converting enzyme gene and ocular signs of primary open-angle glaucoma. Graefes Arch Clin Exp Ophthalmol.; 243(4):294; Costagliola et al., 2000, Effect of oral losartan potassium administration on intraocular pressure in normotensive and glaucomatous human subjects. Exp Eye Res 71(2):167; Costagliola et al., 1995. Effect of oral captopril (SQ 14225) on intraocular pressure in man. Eur J Ophthalmol., 5(1):19; Cullinane et al., 2002, Renin-angiotensin system expression and secretory function in cultured human ciliary body non-pigmented epithelium. Br J Ophthalmol., 86(6):676; Sakaguchi et al., 2002, Chymase and angiotensin converting enzyme activities in a hamster model of glaucoma filtering surgery. Curr Eye Res. 24(5):325; Shah et al., 2000, Oculohypotensive effect of angiotensin-converting enzyme inhibitors in acute and chronic models of glaucoma. J Cardiovasc Pharmacol., 36(2):169, and Wang et al., 2005, Effect of CS-088, an angiotensin AT1 receptor antagonist, on intraocular pressure in glaucomatous monkey eyes. Exp Eye Res., 80(5):629.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNA). After the discovery of the phenomenon in plants in the early 1990s, Andy Fire and Craig Mello demonstrated that double-stranded RNA (dsRNA) specifically and selectively inhibited gene expression in an extremely efficient manner in Caenorhabditis elegans (Fire et al., 1998, Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature, 391:806). The sequence of the first strand (sense RNA) coincided with that of the corresponding region of the target messenger RNA (mRNA). The second strand (antisense RNA) was complementary to the mRNA. The resulting dsRNA turned out to be several orders of magnitude more efficient than the corresponding single-stranded RNA molecules (in particular, antisense RNA).
The process of RNAi begins when the enzyme, DICER, encounters dsRNA and chops it into pieces called small-interfering RNAs (siRNA). This protein belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA (see Bosher & Labouesse, 2000, RNA interference: genetic wand and genetic watchdog. Nat Cell Biol, 2000, 2(2):E31, and Akashi et al., 2001, Suppression of gene expression by RNA interference in cultured plant cells. Antisense Nucleic Acid Drug Dev, 11(6):359).
In attempting to utilize RNAi for gene knockdown, it was recognized that mammalian cells have developed various protective mechanisms against viral infections that could impede the use of this approach Indeed, the presence of extremely low levels of viral dsRNA triggers an interferon response, resulting in a global non-specific suppression of translation, which in turn triggers apoptosis (Williams, 1997, Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation. Biochem Soc Trans, 25(2):509; Gil & Esteban, 2000, Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis, 5(2):107-14).
In 2000 dsRNA was reported to specifically inhibit 3 genes in the mouse oocyte and early embryo. Translational arrest, and thus a PKR response, was not observed as the embryos continued to develop (Wianny & Zernicka-Goetz, 2000, Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Bioi, 2(2):70). Research at Ribopharma AG (Kulmbach, Germany) demonstrated the functionality of RNAi in mammalian cells, using short (20-24 base pairs) dsRNA to switch off genes in human cells without initiating the acute-phase response. Similar experiments carried out by other research groups confirmed these results. (Elbashir et al., 2001, RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev, 15(2):188; Caplen et al., 2001, Specific inhibition of gene expression by small double stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA, 98: 9742). Tested in a variety of normal and cancer human and mouse cell lines, it was determined that short hairpin RNAs (shRNA) can silence genes as efficiently as their siRNA counterparts (Paddison et al, 2002, Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 16(8):948). Recently, another group of small RNAs (21-25 base pairs) was shown to mediate downregulation of gene expression. These RNAs, small temporally regulated RNAs (stRNA), regulate timing of gene expression during development in Caenorhabditis elegans (for review see Banerjee & Slack, Control of developmental timing by small temporal RNAs: a paradigm for RNA-mediated regulation of gene expression. Bioessays, 2002, 24(2):119-29 and Grosshans & Slack, 2002, Micro-RNAs: small is plentiful. J Cell Biol, 156(1):17).
Scientists have used RNAi in several systems, including Caenorhabditis elegans, Drosophila, trypanosomes, and other invertebrates. Several groups have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines (specifically in HeLa cells) demonstrating that RNAi is a broadly applicable method for gene silencing in vitro. Based on these results, RNAi has rapidly become a well recognized tool for validating (identifying and assigning) gene function. RNAi employing short dsRNA oligonucleotides will yield an understanding of the function of genes that are only partially sequenced.
As already stated, IOP is maintained by a balance between aqueous humour (AH) production (dependent on sodium transport across a ciliary epithelial bi-layer) and drainage (predominantly through the trabecular meshwork). AH is secreted into the posterior chamber of the eye flowing from the ciliary epithelium, between the iris and the lens, through the pupillary aperture, entering the anterior chamber, and finally flowing radially to the periphery, where it exits predominantly via the canal of Schlemm in the trabecular meshwork (TM), and to a lesser extent through uveoscleral outflow routes (Davson H. The aqueous humour and intraocular pressure. In: Davson's Physiology of the Eye, 5th edn. London, Macmillan Press, 1990: 3-95; Hart W M. Intraocular pressure. In: Hart W M, ed. Adler's Physiology of the Eye. St Louis, Mosby-Year Book Inc, 1992: 248-267).
In peripheral epithelial tissues, sodium and water transport are regulated by corticosteroids and the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) isozymes (11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1), activating cortisol from cortisone, and 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2), inactivating cortisol to cortisone). 11beta-HSD1 is widely expressed, most notably in many glucocorticoid target tissues including liver, adipose tissue, bone, as well as lung, vasculature, ovary and the central nervous system.
11beta-HSD expression has been described in the human eye. 11beta-HSD2 is expressed in the corneal endothelium, whereas 11beta-HSD1 is more widely expressed in the trabecular meshwork, lens epithelium and corneal epithelium (Tomlinson J W. 11 Beta-hydroxysteroid dehydrogenase type 1 in human disease: a novel therapeutic target. Minerva Endocrinol. 2005 March; 30(1):37-46).
11beta-HSD1 but not 11beta-HSD2 has been localized in the human non-pigmented neuroepithelial cells or NPE (Rauz S, Walker E A, Shackleton C H L, Hewison M, Murray P I, Stewart P M. Expression and putative role of 11β-hydroxysteroid dehydrogenase isozymes within the human eye. Invest Ophthalmol Vis Sci 2001; 42:2037-42; Suzuki T, Sasano H, Kaneko C, Ogawa S, Darnel A D, Krozowski Z S. Immunohistochemical distribution of 11β-hydroxysteroid dehydrogenase in human eye. Mol Cell Endocrinol 2001; 173:121-5; Mirshahi M, Nicolas C, Mirshahi A, Hecquet C, d'Hermies F, Faure J P, et al. The mineralocorticoid hormone receptor and action in the eye. Biochem Biophys Res Commun 1996; 219:150-6; Stokes J, Noble J, Brett L, Philips C, Seckl J R, O'Brien C, et al. Distribution of glucocorticoid and mineralocorticoid receptors and 11β-hydroxysteroid dehydrogenases in human and rat ocular tissues. Invest Ophthalmol Vis Sci 2000; 41:1629-38). In situ hibridization defined expression of 11beta-HSD1 in the ciliary epithelium, while RT-PCR analysis of ciliary body tissue confirmed expression of 11beta-HSD1, with additional glucocorticoid receptor and mineralocorticoid receptor (Rauz S, Cheung C M, Wood P J, Coca-Prados M, Walker E A, Murray P I, Stewart P M. Inhibition of 11beta-hydroxysteroid dehydrogenase type 1 lowers intraocular pressure in patients with ocular hypertension. QJM., 2003 July; 96(7):481-90). The enzyme 11beta-HSD1 plays a pivotal role in determining intracellular glucocorticoid concentrations by regenerating, in a reversible reaction, active glucocorticoid (cortisol in humans, corticosterone in rats and mice) from inactive cortisone and 11-dehydrocorticosterone. A high cortisol/cortisone ratio of 14:1 has been documented in aqueous humour, consistent with this local cortisol-generating system (Rauz S, Walker E A, Shackleton C H L, Hewison M, Murray P I, Stewart P M. Expression and putative role of 11β-hydroxysteroid dehydrogenase isozymes within the human eye. Invest Ophthalmol Vis Sci 2001; 42:2037-42).
Oral administration of carbenoxolone (CBX), an inhibitor of 11beta-HSD1, to volunteers in a pilot uncontrolled study, resulted in a decrease of IOP of 17.5%, suggesting that 11beta-HSD1 activity may partly regulate sodium transport across the NPE-pigmented bi-layer, and consequently aqueous humour secretion (Rauz S, Walker E A, Shackleton C H L, Hewison M, Murray P I, Stewart P M. Expression and putative role of 11β-hydroxysteroid dehydrogenase isozymes within the human eye. Invest Ophthalmol Vis Sci 2001; 42:2037-42). Further, randomised, placebo-controlled studies of healthy volunteers and patients with ocular hypertension (raised IOP but no optic neuropathy) assessed the effect of CBX on IOP (Rauz S, Cheung C M, Wood P J, Coca-Prados M, Walker E A, Murray P I, Stewart P M. Inhibition of 11beta-hydroxysteroid dehydrogenase type 1 lowers intraocular pressure in patients with ocular hypertension. QJM., 2003 July; 96(7):481-90).
The preceding is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows, and is not an admission that any of the work described is prior art to the claimed invention.